A significant complication of using a lipid nanoparticle formulation to deliver an immunogenic or immunostimulatory cargo to a mammalian subject is the propensity of such formulations to induce an immune response (e.g., acting as an immunologic adjuvant) within the subject administered such a formulation. In addition to any immunogenic or immunostimulatory role of a formulation cargo, certain lipids have been identified as capable of activating an innate immune response within a subject—cationic lipids, and lipopolysaccharides in particular, have been identified as activators of TLR4-mediated immune responses. Cationic lipids have also been identified as helping to activate TLR3-, TLR7-, TLR8-, and TLR9-mediated immune responses when formulated with nucleic acid cargoes/payloads (e.g., single- and double-stranded nucleic acids and nucleic acid analogs, including RNA and analogs, DNA and analogs, and nucleic acid mimics or other modifications of oligonucleotides (NAs), and peptides, proteins and analogs, and other formulation components, such as PEG, PEG-linked moieties and other polymers, and lipidic components (excipients).
Immunogenic or immunostimulatory properties of lipid formulations have been identified for a number of cargo classes, including peptides, proteins and nucleic acids. For example, dsRNAs have been described to trigger immune responses via at least two mechanisms: (1) dsRNAs of greater than 30 base pairs in length can trigger immunostimulation (Manche et al. Mol. Cell Biol. 12: 5238-42) by a mechanism dependent upon two or more PKR monomers simultaneously binding such a dsRNA molecule (Lemaire et al. J. Mol. Biol. 381: 351-60); and (2) dsRNAs of any length can also trigger immunostimulatory responses in mammalian subjects via MDA5-, TLR3-, OAS1-, RIG-I-, TLR7-, and/or TLR8-mediated interferon activation. Cationic lipids can potentiate these responses (Hagele et al. Nephrology Dialysis Transplantation 2009 24(11):3312-3318). ssDNAs and dsDNAs and analogs, especially those including base modifications necessary for other pharmacological activities (Kandimala et al. Proc Natl Acad Sci USA. 2005 May 10; 102(19):6925-30), can trigger immunostimuluation via TLR9 (Krieg et al. Nature. 374: 546-9; Kreig, Trends Microbiol. 9: 249-52). Such immunologic responses can produce serious side effects in a subject, including fever, chills, hypotension, arrhythmia, liver damage and other harmful or potentially fatal effects, and can be triggered by formulation components, payloads, or both.
Thus, discovery of a means for reducing or blocking the immunostimulatory (e.g., immunologic adjuvant) properties of a formulation comprising an immunogenic or immunostimulatory cargo or pharmacologically necessary component (e.g., a nucleic acid, protein, small drug molecule and/or excipient) while retaining or improving the desired pharmacological activity of the formulation/cargo (e.g., where the cargo is a nucleic acid, protein, active drug molecule or vehicle component, retaining robust target gene inhibitory and/or other pharmacological efficacy and potency of such formulations) would allow for improved use of such immune response reducing formulations in mammalian subjects, including humans.